Minjie Xie

TWIK-1 and TREK-1 Are Potassium Channels Contributing Significantly to Astrocyte Passive Conductance in Rat Hippocampal Slices

Expression of a linear current–voltage (I–V) relationship (passive) K+ membrane conductance is a hallmark of mature hippocampal astrocytes. However, the molecular identifications of the K+ channels underlying this passive conductance remain unknown. We provide the following evidence supporting significant contribution of the two-pore domain K+ channel (K2P) isoforms, TWIK-1 and TREK-1, to this conductance. First, both passive astrocytes and the cloned rat TWIK-1 and TREK-1 channels expressed in CHO cells conduct significant amounts of Cs+ currents, but vary in their relative PCs/PK permeability, 0.43, 0.10, and 0.05, respectively. Second, quinine, which potently inhibited TWIK-1 (IC50 = 85 μm) and TREK-1 (IC50 = 41 μm) currents, also inhibited astrocytic passive conductance by 58% at a concentration of 200 μm. Third, a moderate sensitivity of passive conductance to low extracellular pH (6.0) supports a combined expression of acid-insensitive TREK-1, and to a lesser extent, acid-sensitive TWIK-1. Fourth, the astrocyte passive conductance showed low sensitivity to extracellular Ba2+, and extracellular Ba2+ blocked TWIK-1 channels at an IC50 of 960 μm and had no effect on TREK-1 channels. Finally, an immunocytochemical study showed colocalization of TWIK-1 and TREK-1 proteins with the astrocytic markers GLAST and GFAP in rat hippocampal stratum radiatum. In contrast, another K2P isoform TASK-1 was mainly colocalized with the neuronal marker NeuN in hippocampal pyramidal neurons and was expressed at a much lower level in astrocytes. These results support TWIK-1 and TREK-1 as being the major components of the long-sought K+ channels underlying the passive conductance of mature hippocampal astrocytes.

Neural cell cycle dysregulation and central nervous system diseases

The cell cycle is a delicately manipulated process essential for the development, differentiation, proliferation and death of cells. Inappropriate activation of cell cycle regulators is implicated in the pathophysiology of a wide range of central nervous system (CNS) diseases, including both acute damage and chronic neurodegenerative disorders. Cell cycle activation induces the dividing astrocytes and microglia to activate and proliferate in association with glial scar formation and inflammatory factor production, which play crucial roles in the development of pathology in CNS diseases. On the other hand, in terminally differentiated neurons, aberrant re-entry into the cell cycle triggers neuronal death instead of proliferation, which may be a common pathway shared by some acquired and neurodegenerative disorders, even though multiple pathways of the cell cycle machinery are involved in distinct neuronal demise in specific pathological circumstances. In this paper, we first provide a concise description of the roles of cell cycle in neural development. We then focus on how neural cell cycle dysregulation is related to CNS diseases. Neuronal apoptosis is often detected in acute injury to the CNS such as stroke and trauma, which are usually related to the blockade of the cell cycle at the G1-S phase. In neurodegenerative diseases such as Alzheimers disease, Parkinsons disease, amyotrophic lateral sclerosis and Niemann-Pick disease type C, however, some populations of neurons complete DNA synthesis but the cell cycle is arrested at the G2/M transition. This review summarizes advances in findings implicating cell cycle machinery in neuronal death in CNS diseases.

Inhibiting cell cycle progression reduces reactive astrogliosis initiated by scratch injury in vitro and by cerebral ischemia in vivo.

Astrogliosis occurs in a variety of neuropathological disorders and injuries, and excessive astrogliosis can be devastating to the recovery of neuronal function. In this study, we asked whether reactive astrogliosis can be suppressed in the lesion area by cell cycle inhibition and thus have therapeutic benefits. Reactive astrogliosis induced in either cultured astrocytes by hypoxia or scratch injury, or in a middle cerebral artery occlusion (MCAO) ischemia model were combined to address this issue. In the cultured astrocytes, hypoxia induced a cell cycle activation that was associated with upregulation of the proliferating cell nuclear marker (PCNA). Significantly, the cell cycle inhibitor, olomoucine, inhibited hypoxia‐induced cell cycle activation by arresting the cells at G1/S and G2/M in a dose‐dependent manner and also reversed hypoxia‐induced upregulation of PCNA. Also in the cultured astrocytes, scratch injury induced reactive astrogliosis, such as hypertrophy and an increase in BrdU(+) astrocytes, both of which were ameliorated by olomoucine. In the MCAO ischemia mouse model, dense reactive glial fibrillary acidic protein and PCNA immunoreactivity were evident at the boundary zone of focal cerebral ischemia at days 7 and 30 after MCAO. We found that intraperitoneal olomoucine administration significantly inhibited these astrogliosis‐associated changes. To demonstrate further that cell cycle regulation impacts on astrogliosis, cyclin D1 gene knockout mice (cyclin D1−/−) were subjected to ischemia, and we found that the percentage of Ki67‐positive astrocytes in these mice was markedly reduced in the boundary zone. The number of apoptotic neurons and the lesion volume in cyclin D1−/− mice also decreased as compared to cyclin D1+/+ and cyclin D1+/− mice at days 3, 7, and 30 after local cerebral ischemia. Together, these in vitro and in vivo results strongly suggest that astrogliosis can be significantly affected by cell cycle inhibition, which therefore emerges as a promising intervention to attenuate reactive glia‐related damage to neuronal function in brain pathology.

Suppression of astroglial scar formation and enhanced axonal regeneration associated with functional recovery in a spinal cord injury rat model by the cell cycle inhibitor olomoucine

It is well established that axons of the adult mammalian CNS are capable of regrowing only a limited amount after injury. Astrocytes are believed to play a crucial role in the failure to regenerate, producing multiple inhibitory proteoglycans, such as chondroitin sulphate proteoglycans (CSPGs). After spinal cord injury (SCI), astrocytes become hypertrophic and proliferative and form a dense network of astroglial processes at the site of lesion constituting a physical and biochemical barrier. Down‐regulations of astroglial proliferation and inhibitory CSPG production might facilitate axonal regeneration. Recent reports indicated that aberrant activation of cell cycle machinery contributed to overproliferation and apoptosis of cells in various insults. In the present study, we sought to determine whether a cell cycle inhibitior, olomoucine, would decrease neuronal cell death, limit astroglial proliferation and production of inhibitory CSPGs, and eventually enhance the functional compensation after SCI in rats. Our results showed that up‐regulations of cell cycle components were closely associated with neuronal cell death and astroglial proliferation as well as the production of CSPGs after SCI. Meanwhile, administration of olomoucine, a selective cell cycle kinase (CDK) inhibitor, has remarkably reduced the up‐regulated cell cycle proteins and then decreased neuronal cell death, astroglial proliferation, and accumulation of CSPGs. More importantly, the treatment with olomoucine has also increased expression of growth‐associated proteins‐43, reduced cavity formation, and improved functional deficits. We consider that suppressing astroglial cell cycle in acute SCIs is beneficial to axonal growth. In the future, therapeutic strategies can be designed to achieve efficient axonal regeneration and functional compensation after traumatic CNS injury.

Cell cycle inhibition attenuates microglia induced inflammatory response and alleviates neuronal cell death after spinal cord injury in rats

The spinal cord is well known to undergo inflammatory reactions in response to traumatic injury. Activation and proliferation of microglial cells, with associated proinflammatory cytokines expression, plays an important role in the secondary damage following spinal cord injury. It is likely that microglial cells are at the center of injury cascade and are targets for treatments of CNS traumatic diseases. Recently, we have demonstrated that the cell cycle inhibitor olomoucine attenuates astroglial proliferation and glial scar formation, decreases lesion cavity and mitigates functional deficits after spinal cord injury (SCI) in rats [Tian, D.S., Yu, Z.Y., Xie, M.J., Bu, B.T., Witte, O.W., Wang, W., 2006. Suppression of astroglial scar formation and enhanced axonal regeneration associated with functional recovery in a spinal cord injury rat model by the cell cycle inhibitor olomoucine. J. Neurosci. Res. 84, 1053-1063]. Whether neuroprotective effects of cell cycle inhibition are involved in attenuation of microglial induced inflammation awaits to be elucidated. In the present study, we sought to determine the influence of olomoucine on microglial proliferation with associated inflammatory response after spinal cord injury. Tissue edema formation, microglial response and neuronal cell death were quantified in rats subjected to spinal cord hemisection. Microglial proliferation and neuronal apoptosis were observed by immunofluorescence. Level of the proinflammatory cytokine interleukin-1beta (IL-1beta) expression in the injured cord was determined by Western blot analysis. Our results showed that the cell cycle inhibitor olomoucine, administered at 1 h post injury, significantly suppressed microglial proliferation and produced a remarkable reduction of tissue edema formation. In the olomoucine-treated group, a significant reduction of activated and/or proliferated microglial induced IL-1beta expression was observed 24 h after SCI. Moreover, olomoucine evidently attenuated the number of apoptotic neurons after SCI. Our findings suggest that modulation of microglial proliferation with associated proinflammatory cytokine expression may be a mechanism of cell cycle inhibition-mediated neuroprotections in the CNS trauma.

Tamoxifen attenuates inflammatory-mediated damage and improves functional outcome after spinal cord injury in rats

Tamoxifen has been found to be neuroprotective in both transient and permanent experimental ischemic stroke. However, it remains unknown whether this agent shows a similar beneficial effect after spinal cord injury (SCI), and what are its underlying mechanisms. In this study, we investigated the efficacy of tamoxifen treatment in attenuating SCI‐induced pathology. Blood–spinal cord barrier (BSCB) permeability, tissue edema formation, microglial activation, neuronal cell death and myelin loss were determined in rats subjected to spinal cord contusion. The results showed that tamoxifen, administered at 30 min post‐injury, significantly decreased interleukin‐1β (IL‐1β) production induced by microglial activation, alleviated the amount of Evans blue leakage and edema formation. In addition, tamoxifen treatment clearly reduced the number of apoptotic neurons post‐SCI. The myelin loss and the increase in production of myelin‐associated axonal growth inhibitors were also found to be significantly attenuated at day 3 post‐injury. Furthermore, rats treated with tamoxifen scored much higher on the locomotor rating scale after SCI than did vehicle‐treated rats, suggesting improved functional outcome after SCI. Together, these results demonstrate that tamoxifen provides neuroprotective effects for treatment of SCI‐related pathology and disability, and is therefore a potential neuroprotectant for human spinal cord injury therapy.

Attenuation of astrogliosis by suppressing of microglial proliferation with the cell cycle inhibitor olomoucine in rat spinal cord injury model.

Microglial activation/proliferation and reactive astrogliosis are commonly observed and have been considered to be closely relevant pathological processes during spinal cord injury (SCI). However, the molecular mechanisms underlying this microglial-astroglial interaction are still poorly understood. We showed recently that the continuous injection of the cell cycle inhibitor olomoucine not only markedly suppressed microglial proliferation and associated release of pro-inflammatory cytokines, but also attenuated astroglial scar formation and the lesion cavity and mitigated the functional deficits in rat SCI animal model. In this study, we asked whether microglial activation/proliferation plays an initial role and also necessary in maintaining astrogliosis in SCI model. Our results showed that traumatic induced microglial activation/proliferation precedes astrogliosis, and the up-regulated GFAP expression at both mRNA and protein levels was temporally posterior to the microglial activation. Furthermore, when the cell cycle inhibitor olomoucine was administered only once 1 h post-SCI that should selectively suppress microglial proliferation, the subsequent SCI induced increase in GFAP expression at 1, 2 and 4 weeks was significantly attenuated, suggesting that microglial activation/proliferation played an important role for the later onset astrogliosis after SCI. Consistent with the results that microglial proliferation always precedes astroglial proliferation and there is at present no evidence of other astroglial precursors, which as always does not mean that they will not be uncovered by further searching, and in view of the fact that microglial-derived pro-inflammatory cytokines promote astrogliosis as we reported recently, these findings together suggest that by release of cytokines and other soluble products, the early onset microglial activation/proliferation can significantly influence the subsequent development of reactive astrogliosis and glial scar formation in SCI animal model.

Inhibition of EGFR/MAPK signaling reduces microglial inflammatory response and the associated secondary damage in rats after spinal cord injury

BackgroundEmerging evidence indicates that reactive microglia-initiated inflammatory responses are responsible for secondary damage after primary traumatic spinal cord injury (SCI); epidermal growth factor receptor (EGFR) signaling may be involved in cell activation. In this report, we investigate the influence of EGFR signaling inhibition on microglia activation, proinflammatory cytokine production, and the neuronal microenvironment after SCI.MethodsLipopolysaccharide-treated primary microglia/BV2 line cells and SCI rats were used as model systems. Both C225 and AG1478 were used to inhibit EGFR signaling activation. Cell activation and EGFR phosphorylation were observed after fluorescent staining and western blot. Production of interleukin-1beta (IL-1β) and tumor necrosis factor alpha (TNFα) was tested by reverse transcription PCR and ELISA. Western blot was performed to semi-quantify the expression of EGFR/phospho-EGFR, and phosphorylation of Erk, JNK and p38 mitogen-activated protein kinases (MAPK). Wet-dry weight was compared to show tissue edema. Finally, axonal tracing and functional scoring were performed to show recovery of rats.ResultsEGFR phosphorylation was found to parallel microglia activation, while EGFR blockade inhibited activation-associated cell morphological changes and production of IL-1β and TNFα. EGFR blockade significantly downregulated the elevated MAPK activation after cell activation; selective MAPK inhibitors depressed production of cytokines to a certain degree, suggesting that MAPK mediates the depression of microglia activation brought about by EGFR inhibitors. Subsequently, seven-day continual infusion of C225 or AG1478 in rats: reduced the expression of phospho-EGFR, phosphorylation of Erk and p38 MAPK, and production of IL-1β and TNFα; lessened neuroinflammation-associated secondary damage, like microglia/astrocyte activation, tissue edema and glial scar/cavity formation; and enhanced axonal outgrowth and functional recovery.ConclusionsThese findings indicate that inhibition of EGFR/MAPK suppresses microglia activation and associated cytokine production; reduces neuroinflammation-associated secondary damage, thus provides neuroprotection to SCI rats, suggesting that EGFR may be a therapeutic target, and C225 and AG1478 have potential for use in SCI treatment.

Effectiveness of strengthened stimulation during acupuncture for the treatment of Bell palsy: a randomized controlled trial

Background: The traditional Chinese theory of acupuncture emphasizes that the intensity of acupuncture must reach a threshold to generate de qi, which is necessary to achieve the best therapeutic effect. De qi is an internal compound sensation of soreness, tingling, fullness, aching, cool, warmth and heaviness, and a radiating sensation at and around the acupoints. However, the notion that de qi must be achieved for maximum benefit has not been confirmed by modern scientific evidence. Methods: We performed a prospective multicentre randomized controlled trial involving patients with Bell palsy. Patients were randomly assigned to the de qi (n = 167) or control (n = 171) group. Both groups received acupuncture: in the de qi group, the needles were manipulated manually until de qi was reached, whereas in the control group, the needles were inserted without any manipulation. All patients received prednisone as a basic treatment. The primary outcome was facial nerve function at month 6. We also assessed disability and quality of life 6 months after randomization. Results: After 6 months, patients in the de qi group had better facial function (adjusted odds ratio [OR] 4.16, 95% confidence interval [CI] 2.23–7.78), better disability assessment (differences of least squares means 9.80, 95% CI 6.29–13.30) and better quality of life (differences of least squares means 29.86, 95% CI 22.33–37.38). Logistic regression analysis showed a positive effect of the de qi score on facial-nerve function (adjusted OR 1.07, 95% CI 1.04–1.09). Interpretation: Among patients with Bell palsy, acupuncture with strong stimulation that elicited de qi had a greater therapeutic effect, and stronger intensity of de qi was associated with the better therapeutic effects. Trial registration: Clinicaltrials.gov no. NCT00685789.

Tamoxifen alleviates irradiation-induced brain injury by attenuating microglial inflammatory response in vitro and in vivo

Irradiation-induced brain injury, leading to cognitive impairment several months to years after whole brain irradiation (WBI) therapy, is a common health problem in patients with primary or metastatic brain tumor and greatly impairs quality of life for tumor survivors. Recently, it has been demonstrated that a rapid and sustained increase in activated microglia following WBI led to a chronic inflammatory response and a corresponding decrease in hippocampal neurogenesis. Tamoxifen, serving as a radiosensitizer and a useful agent in combination therapy of glioma, has been found to exert anti-inflammatory response both in cultured microglial cells and in a spinal cord injury model. In the present study, we investigated whether tamoxifen alleviated inflammatory damage seen in the irradiated microglia in vitro and in the irradiated brain. Irradiating BV-2 cells (a murine microglial cell line) with various radiation doses (2-10 Gy) led to the increase in IL-1 beta and TNF-alpha expression determined by ELISA, and the conditioned culture medium of irradiated microglia with 10 Gy radiation dose initiated astroglial activation and decreased the number of neuronal cells in vitro. Incubation BV-2 cells with tamoxifen (1 microM) for 45 min significantly inhibited the radiation-induced microglial inflammatory response. In the irradiated brain, WBI induced the breakdown of the blood-brain barrier permeability at day 1 post irradiation and tissue edema formation at day 3 post-radiation. Furthermore, WBI led to microglial activation and reactive astrogliosis in the cerebral cortex and neuronal apoptosis in the CA1 hippocampus at day 3 post-radiation. Tamoxifen administration (i.p., 5 mg/kg) immediately post radiation reduced the irradiation-induced brain damage after WBI. Taken together, these data support that tamoxifen can decrease the irradiation-induced brain damage via attenuating the microglial inflammatory response.